The proposed studies will elucidate fundamental mechanisms underlying regulation of neuron glutathione (GSH) homeostasis in response to ethanol (E) and will enable augmentation of these systems to enhance neuroprotection. A key transcription factor regulating GSH homeostasis is Nrf2 which drives requisite transcriptions via binding to the antioxidant response element (ARE). Hypothesis. The central hypothesis is that components of the 3-glutamyl cycle, including internal cellular GSH homeostasis components in neurons, can be up-regulated to protect neurons from E-related apoptotic death. Experiments will define essential mechanisms of ARE-dependent regulation of these components and extend this to interventions that will mitigate E-mediated apoptotic death of neurons. Methods. In vivo studies will utilize rat and mouse binge models. In vitro models will be primary cultures of fetal cerebral cortical neurons. Approaches will include transfections with WT and dominant negative controls, RNA silencing, RT-PCR, luciferase assays, and gel shifts. Specific Aim 1: To address the hypothesis that alleviation of ethanol-related damage to neurons can be achieved by optimizing specific components of neuron GSH homeostasis machinery, at the Nrf2/ARE level. The intent is to utilize the Nrf2/ARE cytoprotective system to prevent E-mediated apoptotic neuron death at two levels; first by increased expression of enzymes controlling GSH synthesis and its reduced state ( 3-glutamyl cysteine ligase, glutathione reductase) and second by enhancing regulation of a GSH scavenging system (;-glutamyl transpeptidase (3GT), and aminopeptidase N. Experiments will define mechanisms of ethanol effects on transcriptional and posttranscriptional events controlling expression of these proteins. They will develop means to mitigate E-induced apoptotic death by manipulating Nrf2/ARE driven transcription at key control points of neuron GSH homeostasis machinery. Specific Aim 2. To determine mechanisms by which ethanol impacts on Nrf2 expression in the neuron. Preliminary studies show that E elicits the up-regulation of Nrf2 protein in neurons which could occur at transcriptional and/or posttranscriptional levels. However, this is insufficient to optimally protect the cells from ethanol-mediated apoptotic death. Thus, we will elucidate how ethanol impacts on Nrf2 expression, with the intent of ultimately enhancing neuroprotective potential. Regulation of Nrf2 will be addressed first by post-translational modifications, Keap1 dependent redox switching, and ubiquitination, and second by regulation at the promoter level. Specific Aim 3. To extend these concepts to the live animal using a well-documented rat model and a transgenic mouse model (Nrf2-/-). Experiments will test the hypothesis that Nrf2 activation in the developing brain enhances protection against E-mediated neuron damage. These studies will determine developmental profiles of controlling components of GSH homeostasis, their responses to E, the role of Nrf2 and 3GT in cytoprotection from E, and means to enhance this neuroprotection. PUBLIC HEALTH RELEVANCE: The intent of the proposed studies is to elucidate fundamental mechanisms underlying neuroprotective regulation of glutathione (GSH) homeostasis in response to ethanol and to develop strategies to enhance these systems to protect neurons from ethanol-mediated apoptotic death and oxidative damage. In response to stressors, an extensive array of cytoprotective genes is activated in neurons, including those which regulate neuron GSH homeostasis. The key transcription factor controlling this response is Nrf2 which drives requisite transcriptions via binding to a cis regulatory unit, the antioxidant response element (ARE). We have shown that ethanol-mediated death of neurons can be blocked by a GSH homeostasis system and preliminary data illustrate that this neuroprotection can be provided within the neuron itself. It is mediated by Nrf2/ARE driven gene expressions which can be controlled. Linterventions will utilize augmentation of the 3-glutamyl cycle or its components which will be manipulated at the molecular level.